Genetic engineering involves inserting new genetic information into existing cells of an organism to create a new genotype in order to modify the organism for the purpose of changing one or more of its characteristics. A living organism may be genetically engineered for a variety of reasons, including incorporating novel and/or beneficial traits into the organism. With regard to crop plants, this may include genetically engineering seeds so that plants that grow from the seeds include one or more beneficial traits. Such genetic engineering may include, but is not limited to, inserting genes encoding a discernable marker or genes conferring resistance to herbicidal compounds. The progeny of individual genetically modified organisms may also contain a genetic element of interest such that researchers may wish to segregate and/or identify organisms (including seeds) that contain such genetic elements of interest from a bulk sample.
Conventional genetic techniques may also be used to combine traits from at least two organisms to produce novel and/or beneficial traits in a “genetic cross” and/or hybrid organism.
It is often difficult to determine whether a particular seed contains a genetic element of interest, especially when mixed with seeds that have not been so modified. Using previous techniques, a particular seed would have to be germinated and then the resulting plant sampled to determine whether the seed might contain the genetic element of interest. Alternatively, the seed itself would have to be sampled.
There are advantages, however, in distinguishing a set of seeds containing a genetic element of interest before the respective plants are germinated, especially in the plant research discipline, where research plot space, personnel for sorting, and time are often limited. In maize and other wind-pollinated crops, plants are frequently hand-pollinated, wherein pollen is manually transferred from the tassel to the silk, and the silk is covered by a shoot bag to prevent pollinated by other plants. This hand pollination process requires significant labor and a tassel bag and shoot bag. The prior elimination of undesired plants (i.e. plants that do not contain a genetic element of interest) by sorting seed may eliminate the need for this labor and time-intensive activity. Additionally, researchers may be interested in the yield potential of the plants germinated from seeds containing the genetic element of interest. Thus, it is important that a statistically-significant number of plants germinate from seeds containing the genetic element of interest in a defined space (i.e. a research plot). Furthermore, researchers desire accuracy and often cannot afford to wait or guess to determine whether plants contain a particular genetic element of interest. Additionally, seed and/or seed tissue sampling is a delicate art. For example, if too much tissue is removed from a particular seed for sampling, there is a risk that the seed may not germinate or produce a viable plant.
Various local, federal, and international regulatory bodies require a high degree of accuracy with respect to the composition of seeds. For example, many regulatory bodies have established seed purity standards that require a “zero-tolerance” policy with regard to seed composition. In such a manner, conventional seed sorting techniques may require a sample of seeds to be re-evaluated through multiple seed sorting passes.
Some processes have been disclosed for visualizing green fluorescent protein (GFP) expression in transgenic plants in order to select transgenic seeds as described, for example, in U.S. Pat. No. 6,947,144 to Kim et al. In particular, the Kim reference describes a system for separating seeds transformed with a green fluorescent protein (GFP) that includes a light source 5′ filtered through a band pass filter 6′. The light source 5′ is positioned above and at a 45° angle from plant samples traveling on a conveyor belt 3′. A CCD camera 9′ is also positioned above the conveyor belt 3′, directly above the examined plant sample 4′. The CCD camera 9′ detects light generated from a portion of the surface area of the plant sample through a filter 7′. See, e.g., the Kim reference, FIG. 9.
However, the system disclosed by the Kim reference suffers from several insufficiencies. For example, there are a number of different traits, including the expression of fluorescent proteins, for which the accurate measurement of a seed in more than a portion of the surface area of the seed may be important. In some instances, the expression of a fluorescent protein may not be uniform across entire surface area of a seed. Thus, if a localized area of the seed expresses the fluorescent protein and that area is facing downward (such as against the conveyor belt in the Kim reference) the CCD camera will not detect the fluorescent protein and the seed will not be correctly sorted.
In addition, the use of GFP for identification and/or selection of transgenic plant material presents several technical challenges. First of all, the excitation and emission wavelengths for GFP visualization are on the fringes of the visible spectrum, and are therefore not easily visible to the eye (or to many conventional visualization systems) using normal “white” light that is present in conventional research and/or manufacturing environments. Furthermore, it has been noted that GFP may result in protein aggregation in vivo in plant material that has been tagged with GFP. It is well-known that unchecked protein aggregation may not only produce adverse effects in seed product, but may also produce unwanted environmental effects if GFP-tagged seeds are introduced into an agricultural environment that interacts with external ecological systems (such as forests and/or wetlands adjacent to an agricultural research plot).
As a result, there is a need for a method and computer program product for distinguishing seeds that contain a marker associated with a selected genotype from a bulk sample of seeds. The method and computer program product should permit a bulk sample to be sorted based on the presence of the marker or the absence of the marker, and should provide a level of convenience, accuracy, speed and environmental safety that is not available using conventional techniques. In some embodiments, the method should provide for improved accuracy in seed sorting, thus substantially avoiding the need for multiple seed sorting passes. Additionally, in some embodiments the method and computer program product should provide the ability to accurately sort a bulk sample of seeds based on the presence or absence of markers associated with a selected genotype using commercially-available high-speed sorting equipment with minimal modifications.